Trypanosoma brucei ssp. are parasitic protozoa responsible for causing human African trypanosomiasis (HAT, sleeping sickness) in humans and nagana in cattle and other livestock. The parasite is endemic to 36 countries in sub-Saharan Africa, where tsetse fly vector control programs have been the major factors in the recent decline of the disease. Still, the disease is invariably fatal when untreated, and the few drugs available to treat HAT have serious limitations such as prolonged treatment regimen, expense, difficult storage requirements, and serious side effects including death. Understanding the basic cell biology of T. brucei is essential to the development of better drugs that will contribute to the WHO goal of total elimination of HAT as a public health problem. One aspect of T. brucei biology that could be exploited for the development of drugs is the trafficking of endocytic cargo. Bloodstream form trypanosomes have an unusually streamlined endomembrane system that allows for very rapid uptake and recycling or degradation of material. Some aspects of this system have been explored, such as the necessity for small Rab GTPases in regulating different compartments. In other eukaryotes, multiple steps in the endocytic pathway are regulated by signaling lipids called phosphoinositides. Phosphatidylinositol can be phosphorylated on positions 3,4, and 5 of the inositol ring to form seven distinct species. Through the localized action of kinases and phosphatases, individual phosphoinositides are restricted to specific membranes. Enrichment of specific phosphoinositides can therefore mark the identity of functional regions or membrane-bound compartments. Downstream functions arise from the subsequent recruitment of effector proteins, which bind to phosphoinositides through conserved domains including PH, PX, and FYVE domains. PI(3)P and PI(3,5)P2 have particularly been implicated in trafficking between endocytic organelles. Despite their importance in other systems, very little is known about phosphoinositides in T. brucei. This proposal seeks to specifically explore the roles of PI(3)P and PI(3,5)P2 in T. brucei endocytic trafficking. We intend to use biosensors to map the subcellular localization of those phosphoinositides by light and electron microscopy. Next, we will use a knockdown approach to disrupt production of PI(3)P and PI(3,5)P2 independently and define their roles in endocytic trafficking. Finally, we will use an affinity purification method t identify potential effector proteins of PI(3)P and PI(3,5)P2. This work will raise many new questions in T. brucei cell biology and potentially lay the groundwork for future drug development exploiting this pathway.